Oncological

Communication

Biosci. Biotech. Res. Comm. 9(3): 428-434 (2016)

Effect of crocin and doxorubicin / radiation on the

breast cancer cell line, Michigan Cancer Foundation-7

Ali Reza Fanayi

, Vahid Changizi

and Majid Safa

Technology of Radiology and Radiotherapy Department, Allied Medical Sciences School,

Tehran University of Medical Sciences, Tehran, Iran

Allied Medical Sciences School, Iran University of Medical Sciences, Tehran, Iran

ABSTRACT

Crocin is the main carotenoid of saffron showing anticancer properties. Doxorubicin as a chemotherapy drug and

X-ray or gamma radiation therapy are used extensively in the treatment of breast cancer. However their side effects

limited their use. The aim of this study was to investigate the apoptosis of Michigan center foundation-7 breast can-

cer cells in monolayer culture (in vitro), using crocin, doxorubicin, radiation, crocin-radiation, and crocin-doxoru-

bicin.To explore the effect of crocin, doxorubicin and radiation, Michigan center foundation -7 cell line was cultured

and treated with different concentrations of crocin and doxorubicin. MTT assay was used to evaluate the toxicity,

PI  owcytometry was used to evaluate the apoptosis and Western blotting was applied to investigate the protein

expression of p53, PARP, and caspase-7.According to the MTT assay, crocin can decrease growth of Michigan center

foundation-7cell in a dose and time dependent manner. The results of  owcytometry also showed that apoptosis rate

was signi cantly higher in the combined test than Separate tests. Western blot analysis also revealed that the proteins

expression in combined groups was much than separated groups. This study revealed the expression of apoptotic

proteins in the combined therapy of saffron and radiation or saffron and drug was signi cantly higher than that in

using radiation or drug alone.

KEY WORDS: CROCIN, DOXORUBICIN, GAMMA RAY, APOPTOSIS, BREAST CANCER

428

ARTICLE INFORMATION:

*Corresponding Author: changizi@sina.tums.ac.ir

Received 17

July, 2016

Accepted after revision 20

Aug, 2016

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2015: 3.48 Cosmos IF : 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

Ali Reza Fanayi et al.

Introduction

Cancer is the second leading cause of death in the world.

In recent years its rate has grown even more than twice.

Breast cancer is the most common cancer (23% of all

cancers) with the highest mortality (16%) among all

women’s’ malignancies. Although the incidence of can-

cer is low in Asia, but there is more abundance of death

due to cancer in most of the Asian countries than that

in Western countries. In Iran breast cancer has the most

incidences among all malignancies in women. In previ-

ous studies it has been found that the incidence of breast

cancer in Iran is lower than that in developed coun-

tries. However, it is still considered as the most common

cancer in Iranian women and there have increased in

incidence in two past decades (Harirchi, Kolahdoozan

et al. 2011).

According to the report of World Health Organiza-

tion, breast cancer is increasing in developing countries

based on some factors such as increase of life expec-

tancy, urbanization and following up the western life

style. In spite of using three important methods for

breast cancer treatment including surgery, chemother-

apy and radiotherapy, it has high mortality rate indi-

cating the inef ciency of these therapeutic methods

(Møller, Wallin et al. 1996).Saffron is commonly con-

sumed in different parts of the world as a medicinal

drug to treat several health disorders(Schmidt, Betti et al.

2007).

Crocin is the main carotenoid of saffron showing anti-

tumor properties (Jaliani, Riazi et al. 2013). Currently, it

has been proved clinically that crocin may improve the

anti-tumor effect as well as reduce the doses of chemo-

therapeutic agents(Naghizadeh, Boroushaki et al. 2008).

Noticeably, crocin inhibits the growth of cancer cells

signi cantly with no side effects on normal cells (Sun,

Xu et al. 2013 and Tazhibi and Feizi 2014) .

Doxorubicin is an Anthracycline drug, which is tra-

ditionally used for breast cancer treatment. Anthracy-

clines induce double-stranded DNA break associated

with the production of free radicals. The free radicals

could lead to inhibition of mitochondrial respiratory

chain enzymes and oxidation of membrane lipids. At

the cellular level, doxorubicin perches between two

nitrogen bases of double stranded DNA helix, causing

inhibition in DNA polymerase and DNA-dependent RNA

polymerase function. This function can lead to suppres-

sion of DNA and RNA synthesis anddamages DNA repair

mechanism. Doxorubicin also changes Topoisomerase II

activity. Doxorubicin has a stronger in uence on the

S phase of the cell. However some problems such as

hematopoietic suppression, nausea, vomiting, extrava-

sation, alopecia and cardiotoxicity lead doxorubicin’s

success to become incomplete (Czeczuga-Semeniuk,

Wołczynski et al. 2004, Howe Luzhna and Kovalchuk

2010 and Octavia, Tocchetti et al. 2012).

Ionizing radiation has ability to change the organic

bases of the nucleotides, the sugar part of DNA mol-

ecule and most importantly cut the DNA molecule(Scott

and Pandita 2006). Studies show that even a small

number of double stranded DNA breaks after the expo-

sure of ionizing radiation could be associated with

cell death.DNA is can damage from ionizing radiation

such as Radiotherapy because it includes informa-

tion to encode biomolecules then single strand break

(SSB), double strand break(DSB), and dimerization could

threat the cell viability strongly. Therefore, the ioniz-

ing radiations have potential to induce DNA damage

and apoptosis.( Criswell, Leskov et al. 2003 Sun, Zhao,

2013)

Apoptosis is a type of common cell death in eukary-

otes.This process is performed during embryonic stage

and tumor suppression (Nicoletti, Migliorati et al. 1991).

The aim of this study was to investigate the apopto-

sis of breast cancer cells (Michigan center foundation-7

cell line) in monolayer culture ( in vitro), using crocin,

doxorubicin, radiation, crocin- radiation and crocin-

doxorubicin.

MATERIAL AND METHODS

CELL LINES AND REAGENTS

The human breast cancer cell lines MICHIGAN CANCER

FOUNDATION-7, a non-invasive estrogen receptor (ER)

positive, were obtained from Cell Bank of the Pasteur

Institute (Tehran, IRAN). Trypsin and crocin were pur-

chased from Sigma. 3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyl tetrazolium (MTT) and Propidium iodide (PI),

purchased from Sigma. Cells were cultured in Dulbecco’s

Modi ed Eagle’s medium (DMEM) high glucose with 5%

fetal bovine serum (Gibco). Doxorubicin was prepared

from Sobhan Darou (Tehran, Iran). Rabbit monoclonal

antibody of caspase-7, caspase-9, PARP and mouse

monoclonal antibody of p53 were bought from Cell

Signaling (USA).

CELL CULTURE AND RESEARCH METHODS

Michigan center foundation-7 cells were cultured in the

DMEM high glucose medium, supplemented with 10%

heat-inactivated fetal bovine serum and maintained in a

humidi ed atmosphere at 370C and 5% CO2.Cellsin the

logarithmic growth period were selected for experimen-

tal studies.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF CROCIN AND DOXORUBICIN / RADIATION ON THE BREAST CANCER CELL LINE 429

Ali Reza Fanayi et al.

MEASUREMENT OF THE SURVIVAL RATES OF

MICHIGAN CENTER FOUNDATION-7 CELLS

WITH MTT METHOD

25000 cells were seeded in the 24-well plate with 1ml

of culture medium for 24 h. Different concentrations

of 1.5mg/ml, 2.5mg/ml, 3.5mg/ml, 4.5mg/ml and 6mg/

ml for the crocin and 0.01M/ml, 0.03 M/ml,0.05M/

ml, 0.1M/ml, 0.5M/ml and 1M/ml for the doxoru-

bicin were examined separately to get IC50 (Inhibition

concentration) for Michigan center foundation-7 at the

minimum possible time. On this base the incubation

times for crocin were used 24 h, 48 h and 72 h and for

doxorubicin 48 h. Then the medium was being removed

and cells were incubated with 100 l (5mg / ml dissolved

in PBS) MTT and 900 l medium culture at 370C for 4

h. For each well the supernatant was discarded, 500ul

DMSO was added and the mixture was suspended. The

light absorbance (A) was measured at 570 nm wave-

length using ELISA. Finally survival rate calculated as

follows: survival rate of tumor cells (%) = experimental

group A value/control group A value×100%.

All above steps were done for crocin and doxorubicin

separately. As a result concentrations of 2.5mg/ml for

crocin and 0.01M/ml for doxorubicin were selected as

the optimized combination. Then four groups of Michi-

gan center foundation-7 were selected to study three

methods of treatments as follows: a control group, sec-

ond group irradiated by 2Gy Gamma radiation (with

cobalt source), and the third group received 2.5mg/ml

crocin with 0.01M/ml doxorubicin and  nally fourth

group was studied by 2.5mg/ml crocin with 2Gy Gamma

radiation. The experiment times were 24 h, 48 h and 72

h for crocin groups and 48 h for another group, after

the treatment with drug. All trials were repeated three

times.

DETERMINATION OF CELL APOPTOSIS BY

FLOWCYTOMETRY

Apoptotic cells were revealed by the  owcytometry

using PI staining to detect the so-called sub-G1 peak

(Riccardi and Nicoletti 2006).This process leads to meas-

ure DNA content after staining nucleic acid with spe-

ci c  uorochromes(Riccardi and Nicoletti 2006) For

this assay MICHIGAN CANCER FOUNDATION-7 cells

were cultured in a 6-well plate (70000 cells per well)

and treated with 2.5mg/ml crocin for 24 h, 48 h and 72

h. The second group of cells was treated with .01M/

ml of doxorubicin and the third group was irradiated

with 2 Gy gamma. To evaluate the combined therapy,

the fourth group was treated by 2.5mg/ml crocin and

0.01 µM/ml doxorubicin for 48 h and the  fth group

was treated by 2 Gy gamma and 2.5mg/ml crocin for 24

incubation. Then for all groups beside the control group

the  ow cytometric analysis was being done.

WESTERN BLOT TO DETECT THE EXPRESSION

OF CASPASE-7 AND CASPASE-9 AND

P53 AND PARP OF MICHIGAN CENTER

FOUNDATION-7 CELLS

Michigan center foundation-7 cell lines were classi-

 ed and treated similar to two pervious methods. Then

treated cells were detached by trypsinization, washed

with PBS, centrifuged 4000 rpm for 5 minutes added

with RIPA (lysis buffer). In next step all samples were

put in ice for 30 minutes and vortexed every 5 minutes

until those would have been homogenized well. Then

samples were centrifuged 13000 rpm for 20 minutes at

C. The supernatant was taken out and the concentra-

tion of protein was being measured by Bradford method.

Protein samples were divided into smaller amounts and

kept at -80

To do western blot at  rst running buffer and trans-

fer buffer were prepared and three steps were followed

as: a) proteins were separated according to molecular

weight by gel electrophoresis, b) transferred to nitrocel-

lulose membrane, c) then the desired protein was speci-

 ed with the primary antibody and shown with the sec-

ondary antibody. Finally antibody bonds appeared with

ECL on the  lm. The thickness of each bond was directly

related to the amount of protein.

RESULTS

CHANGES IN SURVIVAL RATES OF MICHIGAN

CENTER FOUNDATION-7 CELLS

This study revealed the cell growth of MICHIGAN CAN-

CER FOUNDATION-7 cell line was inhibited by crocin in

a dose and time dependent manner (Figure 1 A-C). There

were signi cant differences among different groups (P

value <0.05). IC50 was shown for 3.5 mg/ml crocin in 48

h by MTT (Figure 1 B).The resultes of Crocin MTT asseys

are similar to results of Vali and changizi 2015.

Different concentrations of doxorubicin for 48 incu-

bation could suppress MICHIGAN CANCER FOUNDA-

TION-7 cell line in a dose dependent manner (P value

<0.05) (Fig.1D). For this drug IC50 was obtained 0.1 µM/

ml in 48 h by MTT assay. This research showed com-

bination of different concentrations of crocin and 0.01

µM/ml doxorubicin, may have stronger inhibition effect

than the single agent on breast cancer cells. Similarly it

was shown with combination of 2.5mg/ml of crocin and

430 EFFECT OF CROCIN AND DOXORUBICIN / RADIATION ON THE BREAST CANCER CELL LINE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF CROCIN AND DOXORUBICIN / RADIATION ON THE BREAST CANCER CELL LINE 431

Ali Reza Fanayi et al.

2Gy radiation the survival rate was signi cantly lower

(P value <0.05) (Fig. 1E _

APOPTOTIC CHANGES OF MICHIGAN CANCER

FOUNDATION-7 CELLS

Apoptosis following treatment with crocin, doxorubicin,

radiation and combination of them was measured by

 owcytometry using PI staining to detect the sub-G1

peak resulting from DNA fragmentation. The results

showed apoptosis rate with crocin to be increased with

duration of time (P < 0.05) (Fig. 2 A _ D). Also this study

revealed the combined treatment of crocin and doxoru-

bicin or crocin and radiation have more apoptotic effect

than the single agent (P < 0.05) (Fig. 2 E _ H).

EXPRESSION OF CASPASE-9, CASPASE-7,

P53 AND PARP IN MICHIGAN CANCER

FOUNDATION-7 CELLS

With exposure of 2.5 mg/ml and 4.5 mg/ml crocin to

MICHIGAN CANCER FOUNDATION-7 cells for 48h,

FIGURE 1: The inhibition effect of crocin, Doxorubicin, Radiation and com-

bination of them on MICHIGAN CANCER FOUNDATION-7 cells, measured by

MTT (A) crocin 24h, (B) crocin 48h, (C) crocin 72 h. (D) Doxorubicin 48h (E)

combination of crocin in all concentration 48h with 0.01uM/ml Doxorubicin

48h (F) combination of crocin 48h with radiation 24h.

FIGURE 2: Flowcytometry histograms of PI-stained MICHIGAN CANCER FOUN-

DATION-7 cells and Sub-G1 peak means apoptotic cells.(A) control 48h (B)

Crocin 2.5 mg/ml 24h (C) Crocin 2.5 mg/ml 48h (D) crocin 2.5 mg/ml 72h (E)

doxorubicin .01uM/ml 48h (F) crocin 2.5mg/ml 48h + doxorubicin .01uM/ml (G)

radiation 2Gy (H) crocin 2.5 mg/ml 48h + radiation 2Gy 24h.

432 EFFECT OF CROCIN AND DOXORUBICIN / RADIATION ON THE BREAST CANCER CELL LINE BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Ali Reza Fanayi et al.

expression of p53, PARP, caspase-9 and caspase-7 was

detected. The results showed that the expression of all

four proteins was in a dose dependent manner (Fig. 3 A).

Also expression of these proteins was evaluated in com-

bination of crocin with doxorubicin or radiation. The

results showed that the expression of apoptotic proteins

in the combined treatments were signi cantly higher

than the single groups (Fig. 3 B_C).

DISCUSSION

Cancer is the second leading cause of death in the world.

In recent years its rate has been grown even more than

twice. Breast cancer is the most common cancer (23%

of all cancers) with the highest mortality (16%) among

all malignancies in women. Therefore breast cancer

remains one of the main health problem in the word

(Harirchi, Kolahdoozan et al. 2011). Studies show that

increased incidence of the breast cancer is higher in

developing countries and life expectancy of patients is

lower (Shibuya, Mathers et al. 2002). The reasons of can-

cer are associated with the environmental factors such

as air pollution, stress, diet and life style of the people. It

has been found that the consumption of foods that have

antioxidant properties, is effective in reducing the risk of

cancer (Ren, Qiao et al. 2003).

On the other hand, despite the use of therapeutic

options including surgery chemotherapy and radiother-

apy for patients mortality remains high. It indicates the

inef ciency of these ways. Moreover destructive effects

of chemotherapy and radiation on normal cell division

are also other disadvantages associated with the thera-

peutic process (Chabner Ba Fau - Friedman and Fried-

man). For example, while doxorubicin is a valuable anti-

cancer agent, cardiotoxicity and drug resistance are two

main problems of this drug (Kaye and Merry 1985, Weiss

1992).

Crocin is the main carotenoid of saffron with anti-

tumor properties (Jaliani, Riazi et al. 2013). Study

on HepG2 cell line showed telomerase activity in the

nucleus would decrease with increasing concentration

of crocin and it would lead to inhibition of prolifera-

tion and apoptosis (Noureini and Wink 2012). Our study

could approve this result. It was also found that DNA

fragmentation and cell cycle arrest are the main signs

of apoptosis in pancreatic cancer cells treated with

crocin(Bakshi, Sam et al. 2010). Gupta and colleagues in

October 2014 investigated the synergistic effect of crocin

and cisplatin on MDA MB-231 cells and MCF_7. It was

found that lower concentrations of cisplatin along with

crocin can achieve the desired result (Gupta, Jhamb et al.

2014). Notably, crocin signi cantly inhibits the growth

of cancer cells with no effects on normal cells (Sun, Xu

et al. 2013).

Consistent with the results of other studies, the results

of this study also con rms the effectiveness of crocin

against breast cancer Michigan center foundation-7 cell

line. To evaluate the survival rate of Michigan center

foundation-7 cells in treatment with doxorubicin, crocin,

radiation and combined therapy MTT assay was used.

This study showed treatment of Michigan center foun-

dation -7 cells with crocin was time and dose-dependent

to decrease the rate of proliferation. For example when

the concentration of crocin increased from 1.5 mg/ml to

6mg/ml in 48h, the survival rate of MCF-7 cells reduced

from 75% to 23%. Also the survival rate of MCF-7cells

reduced from 75% to 22% in treatment of cells with

doxorubicin from .01 µM/ml to 1µM/ml in 48h. It means

doxorubicin reduces the survival rate in a dose depend-

ent manner.

Cellular stresses such as ionizing radiation, UV and

chemical carcinogens can activate p53, including dam-

age to DNA, oncogene expression, hypoxia and nucleo-

tide depletion (Giaccia and Kastan 1998). According to

the type and severity of toxicity, p53 protein causes cell

cycle arrest or cell death through apoptosis that the for-

mer cause to repair DNA and the second led to remove

from cell replication(Bouvard, Zaitchouk et al. 2000) . P53

causes to release cytochrome C from mitochondria and

induces the expression of Apaf-1 forming the apopto-

FIGURE 3: Evaluating the expression of caspase-9, caspase-7, p53 and PARP (A)

in a concentration of 2.5 and 4.5 mg/ml in 48 h. (B) in dox .01uM/ml , crocin

2.5mg/ml + dox.01uM/ml (c) in radiation 2Gy , crocin2.5+radiation2G.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS EFFECT OF CROCIN AND DOXORUBICIN / RADIATION ON THE BREAST CANCER CELL LINE 433

Ali Reza Fanayi et al.

some (Kannan, Kaminski et al. 2001). Apoptosome leads

to activate caspase-9 following with executive caspases

such as 3,6 and 7 (Hengartner 2000). The caspase-acti-

vated DNase cut DNA between nucleosomes (Pecorino).

This process leads to measure DNA content after stain-

ing nucleic acid with speci c  uorochromes(Riccardi

and Nicoletti 2006) . PI is a  uorogenic compound that

binds to nucleic acids, so that  uorescence emission is

proportional to the DNA content of a cell. When apop-

totic cells are stained with PI and analyzed with a  ow

cytometer, they show a wide hypodiploid (sub-G1) peak,

that easily discriminates from narrow peak of normal

cells (diploid cells ) (Riccardi and Nicoletti 2006).

Michigan center foundation is a cancer cell line with

defect in caspase-3 and is relatively insensitive to many

chemotherapy drugs (Yang, Sladek et al. 2001). PARP

as the cellular protein is cleaved speci cally in apopto-

sis. Particular proteolysis of PARP happens in the DNA

binding domain. Caspase-3 and caspase-7 are the most

effective proteases for PARP cleavage (Herceg and Wang

2001). Therefore PI  owcytometry and western blot were

used to evaluate breast cancer cells apoptosis in our study.

Flowcytometry showed to treat MCF 7cells for 48 hours

with 0.01 µmol/ml doxorubicin causes 24.17% apoptosis.

The combined therapy of 2.5 mg/ml crocin and 0.01µm/

ml Doxorubicin for 48 hours with a synergistic effect

caused 50.17% apoptosis. Also the combined therapy

of crocin and gamma radiation with a synergistic effect

could cause 46.60% apoptosis in breast cancer cells.

CONCLUSION

This study revealed the expression of apoptotic proteins

in the combined therapy of saffron and radiation or

saffron and drug was signi cantly higher than that in

using radiation or drug alone. Finally it was found that

crocin could be an appropriate supplement for treatment

of breast cancer by reducing the dosage and harmful

effects of drugs or radiation.

ACKNOWLEDGMENT

This study has been supported by Tehran University of

Medical Sciences. Grant number: 25349

CONFLICT OF INTEREST

There is no con ict of interest

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